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Small vessel diseases in a mechanistic perspective: Targets for Intervention Affected pathways and mechanistic exploitation for prevention of stroke and dementia

Periodic Reporting for period 3 - SVDs-at-target (Small vessel diseases in a mechanistic perspective: Targets for InterventionAffected pathways and mechanistic exploitation for prevention of stroke and dementia)

Reporting period: 2019-01-01 to 2020-06-30

Cerebral small vessel disease (SVD) covers a variety of changes in the small arterioles and venules in the brain. It gives rise to one in five strokes and constitutes a major source of cognitive decline and disability in the elderly. The disease process and mechanisms remain largely unknown and there are no treatments with proven efficacy. This network of basic scientists and academic clinicians elucidates the main risk factors of SVDs: blood pressure variability (BPv), leakage of the blood brain barrier, the role of the extracellular matrix and activation of inflammation.
The main objectives are to:
-define common molecular, cellular, and physiological mechanisms underlying the regulation of blood flow, and barrier and clearance functions of microvessels.
-determine how these common mechanistic defects intersect to drive brain parenchymal damage.
.validate the relevance of mechanisms and biomarkers through interventions.
In the third period, we made substantial progress in all work packages:

Blood Pressure variability and microvascular dysfunction
Functional hyperemia, the blood flow in response to nerve activity, is progressively impaired in a mouse model of chronic hypertension (BPH/2J mice), due to crippled capillary-to-arteriole vasodilatory signaling and capillary Kir2.1 current downregulation. Antihypertensive treatment, employing three clinically used drugs with distinct pharmacological targets, significantly and similarly lowered blood pressure in BPH/2J mice. However, we found that the beneficial effects of antihypertensive treatment on functional hyperemia and capillary-to-arteriole signaling depend on the class of antihypertensives. We are currently deciphering the molecular mechanism(s) causing this class-effect of antihypertensives.

Hypertension causes structural and functional alterations in the vasculature and is the leading risk factor for cerebrovascular diseases in humans. Recent studies indicating the deleterious influence of mid-life hypertension on later-life cognitive function highlight the importance of adequate blood pressure control. In addition, the risk of stroke is higher in those patients with more variable systolic BP at each level of mean systolic BP.

Blood brain barrier (BBB) and perivascular flow
No BBB defects have been identified in hypertensive mice or rats and in CADASIL mice. Two-photon tracking of particles in the perivascular space requires anesthetised mice as the microspheres do not enter the perivascular spaces in awake mice. Application of anaesthesia leads to a decrease of BP, whereas high BP is essential for vascular pulsatility. We currently try to find an agent that restores the high BP in anesthetised BHP/2J to a level comparable with awake mice.

Recruitment in the clinical study INVESTIGATE-SVDs, which uses MRI brain scans to assess microvascular function by measuring BBB permeability, cerebral vascular reactivity (CVR), and cerebral pulsatility are completed. Initial results indicate that CADASIL patients have much worse White Matter Hyperdensities (WMH) but also much worse lacunes, microbleeds and perivascular space visibility than patients with sporadic SVDs. We confirm that BBB leakage increases with WMH severity and that CVR is lower in patients with severe WMH, higher vascular pulsatility and with more visible perivascular spaces.

Microvascular matrisome
From the proteomic analysis, we selected two proteins TIMP3 and COL4A1, the expression of which is changed in brain arteries of SVDs. Excess of TIMP3 impairs functional hyperemia in CADASIL mice by loss of capillary-to-arteriole signalling due to Kir2.1 channel downregulation. Col4a1 mutant mice with downregulated COL4A1 exhibit hypermuscularisation of the transitional segment (TS) at the interface between arterioles and capillaries. This increases the pressure in upstream arterioles, which leads together with a loss of smooth muscle cells to arteriolar rupture. Hypermuscularization of TS was confirmed in humans, representing a general mechanism of genetic and sporadic deep ICH.

The clinical study ZOOM@SVDs aims to establish novel markers of microvascular dysfunction in patients with SVD. We demonstrated in CADASIL patients that cerebral perforating artery flow and pulsatility at 7T MRI are novel SVD markers, which is a breakthrough in SVD research in humans. These are the first MRI markers that directly assess small vessel malfunction directly at the level of the small vessels themselves.

Inflammatory mechanisms
The hypothesis that immune cell extravasation takes place in SVDs was not confirmed. Instead, we found that resident microglia activation is associated with human SVD and mouse SVD models. Depletion of microglial improved cognitive impairment in an animal model supporting the hypothesis that microglia plays a critical role in pathogenesis of cognitive impairment. We will now focus on the microglia activation in human SVD and whether the isolated changes in small artery permeability are relevant to human SVD pathology.

For immune cell characterization of patients with SVDs, we collected blood samples across all studies. Central flow cytometry analysis will start in autumn 2020. Characterised immune cell populations will then be correlated with different SVD MRI markers and clinical outcome.

Validation of mechanisms through intervention
The aim of this study was to explore the possibility of phosphatidylinositol 4,5-bisphosphate (PIP2), a minor phospholipid on plasma membrane, as a novel therapeutic to improve cerebral blood flow regulation in mouse models of SVD. We found that exogenous PIP2 treatment improves functional hyperemia and capillary-to-arteriole vasodilatory signaling in a mouse model of CADASIL, the most common genetic cause of SVD, suggesting candidacy of PIP2 as a future therapeutic strategy.

In the multicentre trial TREAT-SVDs three antihypertensive medications are compared with respect to their beneficial effects on microvascular function in patients with SVDs. Mid-term recruitment was reached and initial analysis revealed that patients with sporadic SVDs show a different vascular risk profile than CADASIL patients. This was expected since CADASIL affects younger patients. Statistical analysis showed a low variance in CVR measurements across all study sites, due to extensive harmonization efforts. Comparisons between sporadic SVDs and CADASIL patients demonstrate that CVR at the end of the wash-out phase is higher in sporadic SVDs patients than in CADASIL patients, which is further in line with INVESTIGATE-SVDs.

So far, the project has published 68 scientific publications.
SVDs@target tackles one of the most pressing health issues in ageing societies. The ambition is to identify key mechanisms of SVDs and to validate novel mechanisms through intervention, with the ultimate goal of reducing the burden of SVDs, stroke and dementia.
Our preliminary results already provide a better understanding of disease pathways leading from basic risk factors to functional deficits. They provide new directions for clinical research, such as the importance of early life BP and BPv for stroke and dementia risk and the role of microglia for cognitive impairment. In addition, two novel MRI markers for microvascular dysfunction in patients with SVD were established.
SVDs@target will improve the preventive treatment of the disease and will lead to a significant benefit at the individual and societal level.